Facultative Air-breathing Fish Research Articles

Gut-immunity modulation in Lepidocephalichthys guntea during Aeromonas hydrophila-infection and recovery assessed with transcriptome data

The fish immune system, which consists of innate and adaptive immunologic processes, defends against viruses, bacteria, fungi, and parasites. The gut immunity is an integral part of the host immune system that controls immunological homeostasis, hosts' interactions with their microbiomes, and provides defence against a number of intestinal infections. Lepidocephalichthys guntea, a facultative air-breathing fish, was experimentally infected with Aeromonas hydrophila using intraperitoneal injection followed by bath challenge, and transcriptome data were used to examine the gut immune responses during disease progression and recovery from the diseased state without the use of medication. For the control or uninfected fish (FGC) and the infected fish that were kept for seven days (FGE1) and fifteen days (FGE2), separate water tanks were set up. Coding DNA sequences (CDS) for FGC and FGE1, FGC and FGE2, and FGE1 and FGE2 were analyzed for differential gene expression (DGE). The presence and expression of genes involved in the T cell receptor (TCR) signalling pathway, natural killer (NK) cell-mediated cytotoxicity pathway, and complement-mediated pathway, along with a large number of other immune-related proteins, and heat shock protein (HSPs) under various experimental conditions and its relationship to immune modulation of the fish gut was the primary focus of this study. Significant up-and-down regulation of these pathways shows that, in FGE1, the fish's innate immune system was engaged, whereas in FGE2, the majority of innate immune mechanisms were repressed, and adaptive immunity was activated. Expression of genes related to the immune system and heat-shock proteins was induced during this host's immunological response, and this information was then used to build a thorough network relating to immunity and the heat-shock response. This is the first study to examine the relationship between pathogenic bacterial infection, disease reversal, and modification of innate and adaptive immunity as well as heat shock response.

Gular Cutaneous Fibrosarcoma and Anatomophysiological Considerations for Anaesthesia in a Saddled Bichir, Polypterus endlicheri endlicheri

Bichirs (Polypterus spp.) have frequently been studied with regard to comparative and developmental anatomy; however, very little information has been published regarding diseases, aging changes, and medical and surgical management in these species. Neoplasia represents one such example of conditions for which a dearth of information in these species exists. There has been increasing recognition of various types of neoplasms, including cutaneous tumors, particularly in ornamental fish; some of which may be related to environmental or to infectious causes. When excision of such tumors is indicated, surgical anaesthesia is required. However, special considerations may be warranted when employing immersion anaesthesia protocols in facultative air-breathing fish that can utilise the lungs for respiration. This anatomophysiological feature of Polypterus spp. may not only influence induction and maintenance of surgical anaesthesia but may theoretically have implications for drowning. Herein, we describe the management of a case of a rapidly growing gular neoplasm in a juvenile saddled bichir and considerations for surgical anaesthesia in this “lunged” species. Induction and maintenance of surgical anaesthesia using tricaine methanesulfonate (MS-222) in this species were found to be effective at significantly lower concentrations than standard recommended doses. Histopathological analysis identified the mass as a fibrosarcoma. To the best of our knowledge, this is the first report of a cutaneous fibrosarcoma in a bichir, representing the first report of neoplasia in Polypterus spp. and the first description of surgical anaesthesia in this amphibious fish.

Cardiac contractility of the African sharptooth catfish, Clarias gariepinus: role of extracellular Ca2+, sarcoplasmic reticulum, and β-adrenergic stimulation.

This study investigated the dependence of contraction from extracellular Ca2+, the presence of a functional sarcoplasmic reticulum (SR), and the effects of β-adrenergic stimulation using isometric cardiac muscle preparations. Moreover, the expression of Ca2+-handling proteins such as SR-Ca2+-ATPase (SERCA), phospholamban (PLN), and Na+/Ca2+ exchanger (NCX) were also evaluated in the ventricular tissue of adult African sharptooth catfish, Clarias gariepinus, a facultative air-breathing fish. In summary, we observed that (1) contractility was strongly regulated by extracellular Ca2+; (2) inhibition of SR Ca2+-release by application of ryanodine reduced steady-state force production; (3) ventricular myocardium exhibited clear post-rest decay, even in the presence of ryanodine, indicating a decrease in SR Ca2+ content and NCX as the main pathway for Ca2+ extrusion; (4) a positive force-frequency relationship was observed above 60 bpm (1.0 Hz); (5) ventricular tissue was responsive to β-adrenergic stimulation, which caused significant increases in twitch force, kept a linear force-frequency relationship from 12 to 96 bpm (0.2 to Hz), and improved the cardiac pumping capacity (CPC); and (6) African catfish myocardium exhibited similar expression patterns of NCX, SERCA, and PLN, corroborating our findings that both mechanisms for Ca2+ transport across the SR and sarcolemma contribute to Ca2+ activator. In conclusion, this fish species displays great physiological plasticity of E-C coupling, able to improve the ability to maintain cardiac performance under physiological conditions to ecological and/or adverse environmental conditions, such as hypoxic air-breathing activity.

Striped catfish (Pangasianodon hypophthalmus) use air-breathing and aquatic surface respiration when exposed to severe aquatic hypercarbia.

We investigated the extent to which the facultative air-breathing fish, the striped catfish (Pangasianodon hypophthalmus), uses air-breathing to cope with aquatic hypercarbia, and how air-breathing is influenced by the experimental exposure protocol and level of hypercarbia. We exposed individuals to severe aquatic hypercarbia (up to Pw CO2 = 81 mmHg) using step-wise and progressive exposure protocols while measuring gill ventilation rate, heart rate, mean arterial blood pressure, and air-breathing frequency, as well as arterial blood pH and PCO2 . We confirm that P. hypophthalmus is tolerant of hypercarbia. Under both protocols gill ventilation rate, heart rate, and mean arterial blood pressure were maintained near control levels even at very high CO2 levels. We observed a marked amount of individual variation in the PwCO2 at which air-breathing was elicited, with some individuals not responding at all. The experimental protocol also influenced the onset of air-breathing. Air-breathing began at lower Pw CO2 in the step-wise protocol (23 ± 4.1 mmHg) compared with the progressive protocol (46 ± 7.8 mmHg). Air-breathing was often followed by aquatic surface respiration, at higher PCO2 (71 ± 5.2 mmHg) levels. On average, the blood PCO2 was approximately 43% lower (46 ± 2.5 mmHg) than water Pw CO2 (~81 mmHg) at our highest tested CO2 level. While this suggests that aerial CO2 elimination is an effective, and perhaps critical, respiratory strategy used by P. hypophthalmus to cope with severe hypercarbia, this observation may also be explained by a long lag time required for equilibration.

Effects of carbon dioxide (CO2) at different temperatures on physiological parameters and growth in striped catfish (Pangasianodon hypophthalmus) juveniles

The striped catfish (Pangasianodon hypophthalmus) is a facultative air-breathing fish that is cultured in freshwater culture systems in Mekong Delta, Viet Nam. We report the effects of increased CO2 concentrations (7, 14, and 21 mmHg) and CO2 at different temperatures (27, 30, and 33 °C) on: (i) acid-base status and stress indicators (plasma glucose and cortisol); and (ii) growth parameters including weight, daily weight gain (DWG), specific growth rate (SGR), food conversion ratio (FCR), and digestive enzyme activity in P. hypophthalmus juveniles. The results show that there were stress responses via increases of glucose and cortisol concentrations, sharp decreases in pHe (extracellular pH) and increases in PaCO2 (partial pressure of CO2). Acid-base regulation appeared at the latter stage of CO2 exposure (from 24 h) at elevated temperatures with a related increase of plasma HCO3−. Digestive enzyme activities tended to reduce, typically pepsin at the high CO2 exposures of 14 and 21 mmHg after 60 days. Although survival rate fluctuated from 95 to 100% for all treatments, the lowest weight (56 g/fish) was found in the treatment of 21 mmHg CO2 whereas the control was 75 g/fish after 3 months of culture. In addition, DWG and SGR were relatively similar between the treatments of 7 mmHg CO2 and 14 mmHg CO2 at all three temperatures, whereas higher CO2 concentration led to significantly higher FCR at all CO2 exposures. In conclusion, there were no significant negative impacts of elevated temperatures (27–33 °C) and CO2 exposures (7–21 mmHg) in isolation and in combination on physiological responses, although there was decreased growth performance at the highest concentration of CO2 (21 mmHg).

Effects of temperature on acid-base regulation, gill ventilation and air breathing in the clown knifefish, Chitala ornata.

Chitala ornata is a facultative air-breathing fish, which at low temperatures shows an arterial PCO2 (PaCO2 ) level only slightly elevated above that of water breathers. By holding fish with in-dwelling catheters at temperatures from 25 to 36°C and measuring blood gasses, we show that this animal follows the ubiquitous poikilotherm pattern of reducing arterial pH with increasing temperature. Surprisingly, the temperature increase caused an elevation of PaCO2 from 5 to 12 mmHg while the plasma bicarbonate concentration remained constant at around 8 mmol l-1 The temperature increase also gave rise to a larger fractional increase in air breathing than in gill ventilation frequency. These findings suggest that air breathing, and hence the partitioning of gas exchange, is to some extent regulated by acid-base status in air-breathing fish and that these bimodal breathers will be increasingly likely to adopt respiratory pH control as temperature rises, providing an interesting avenue for future research.

Impact and tissue metabolism of nitrite at two acclimation temperatures in striped catfish (Pangasianodon hypophthalmus)

Elevated concentrations of nitrite develop occasionally in various aquatic habitats and aquaculture facilities, providing a potential danger for freshwater fish that take up nitrite via the gill chloride uptake mechanism. We studied the uptake, effects and metabolism of nitrite in blood, heart and skeletal muscle at two temperatures in striped catfish Pangasianodon hypophthalmus, a facultative air-breathing fish that is heavily cultivated in Southeast Asia. Exposure to 0.8 mM ambient nitrite increased blood [nitrite] and [methaemoglobin] (metHb) to high values at day 1, but values subsequently decreased towards controls at day 7. Blood [nitrite] and metHb content were unexpectedly higher at 27 °C (∼1.2 mM; 69% at day 1) than at 33 °C (∼0.9 mM; 55%), reflecting a lower nitrite uptake at the highest temperature, possibly via an increased reliance on air-breathing relative to water-breathing with temperature increase. A large fraction of the nitrite taken up was effectively eliminated by being detoxified to nitrate. Further, erythrocyte metHb reductase activity was increased during nitrite exposure, efficiently reducing metHb to functional haemoglobin. The uptake of nitrite into white skeletal musculature (main part of the fish) was much lower than into heart tissue. While heart [nitrite] was close to blood plasma levels, muscle [nitrite] peaked at ∼0.2 mM at day 1 and subsequently declined to ∼0.05 mM at day 7, which is below levels reported in various commercial cured meat products. Nitrite was partly metabolized to iron-nitrosyl, S-nitroso and N-nitroso compounds. The increase in nitros(yl)ated compounds was marginal in skeletal muscle and more pronounced in heart tissue.

Oxygenation properties of hemoglobin and the evolutionary origins of isoform multiplicity in an amphibious air-breathing fish, the blue-spotted mudskipper (Boleophthalmus pectinirostris).

Among the numerous lineages of teleost fish that have independently transitioned from obligate water breathing to facultative air breathing, evolved properties of hemoglobin (Hb)-O2 transport may have been shaped by the prevalence and severity of aquatic hypoxia (which influences the extent to which fish are compelled to switch to aerial respiration) as well as the anatomical design of air-breathing structures and the cardiovascular system. Here, we examined the structure and function of Hbs in an amphibious, facultative air-breathing fish, the blue-spotted mudskipper (Boleophthalmus pectinirostris). We also characterized the genomic organization of the globin gene clusters of the species and we integrated phylogenetic and comparative genomic analyses to unravel the duplicative history of the genes that encode the subunits of structurally distinct mudskipper Hb isoforms (isoHbs). The B. pectinirostris isoHbs exhibit high intrinsic O2 affinities, similar to those of hypoxia-tolerant, water-breathing teleosts, and remarkably large Bohr effects. Genomic analysis of conserved synteny revealed that the genes that encode the α-type subunits of the two main adult isoHbs are members of paralogous gene clusters that represent products of the teleost-specific whole-genome duplication. Experiments revealed no appreciable difference in the oxygenation properties of co-expressed isoHbs in spite of extensive amino acid divergence between the alternative α-chain subunit isoforms. It therefore appears that the ability to switch between aquatic and aerial respiration does not necessarily require a division of labor between functionally distinct isoHbs with specialized oxygenation properties.

The integument of the nonamphibious goby Gobionellus oceanicus: Its functional morphology and respiratory capacity.

Facultative air-breathing fish can exchange respiratory gases using an air-breathing organ (ABO), such as the oral cavity of the integument, during environmental hypoxia. The goby Gobionellus oceanicus inhabits areas subject to environmental hypoxia; however, its ABO is unknown. To investigate the respiratory potential of G. oceanicus, the gill and integument surface area, diffusion capacity, and their diffusion barrier thickness were measured. Our results show that although gill surface area is smaller than observed in other facultative air-breathing fish, but it has all features necessary to perform aquatic gas exchange. Additionally the integument of the palate has a short diffusion barrier thickness and a large calculated O2 -diffusion capacity suggesting that it functions as the ABO.

Ventilatory responses of the clown knifefish, Chitala ornata, to hypercarbia and hypercapnia.

The aim of the present study was to determine the roles of externally versus internally oriented CO2/H+-sensitive chemoreceptors in promoting cardiorespiratory responses to environmental hypercarbia in the facultative air-breathing fish, Chitala ornata (the clown knifefish). Fish were exposed to environmental acidosis (pH ~ 6.0) or hypercarbia (≈ 30torr PCO2) that produced changes in water pH equal to the pH levels of the acidotic water to distinguish the relative roles of CO2 versus H+. We also injected acetazolamide to elevate arterial levels of PCO2 and [H+] in fish in normocarbic water to distinguish between internal and external stimuli. We measured changes in gill ventilation frequency, air breathing frequency, heart rate and arterial blood pressure in response to each treatment as well as the changes produced in arterial PCO2 and pH. Exposure to normocarbic water of pH 6.0 for 1h did not produce significant changes in any measured variable. Exposure to hypercarbic water dramatically increased air breathing frequency, but had no effect on gill ventilation. Hypercarbia also produced a modest bradycardia and fall in arterial blood pressure. Injection of acetazolamide produced similar effects. Both hypercarbia and acetazolamide led to increases in arterial PCO2 and falls in arterial pH although the changes in arterial PCO2/pH were more modest following acetazolamide injection as were the increases in air breathing frequency. The acetazolamide results suggest that the stimulation of air breathing was due, at least in part, to stimulation of internally oriented CO2/H+ chemoreceptors monitoring blood gas changes.

HAEMATOLOGICAL CHANGES IN A FRESH WATER FISH, CHANNA PUNCTATUS UNDER CARTRIZ EXPOSURE

Toxic effects of carbamate pesticide cartriz on the haematological characters in a facultative air breathing fish, Channa punctatus has been investigated in the present study. The intoxication of pesticide exhibited a significant decrease in RBC count, Haemoglobin concentration, packed cell volume (PCV) and oxygen carrying capacity of blood, whereas a progressive rise in total leucocyte count (TLC) and clotting time was observed in varying period of intoxication, revealing toxic nature of the agro chemical.

Using an acoustic telemetry array to assess fish volumetric space use: a case study on impoundments, hypoxia and an air-breathing species (Neoceratodus forsteri)

Facultative air-breathing fish can persist in hypoxic waters due to their capacity to acquire atmospheric oxygen. Most studies examining responses of air-breathing fish to aquatic hypoxia have occurred under experimental conditions. How air-breathing fish respond to hypoxic conditions in the field has received less attention. Using depth sensor transmitters and an array of acoustic receivers to monitor the facultative air-breathing Australian lungfish (Neoceratodus forsteri), we investigated habitat preferences and behavioural responses to seasonal hypoxic zones in a riverine impoundment. Three-dimensional (3-D) kernel utilisation distribution (KUD) models revealed that during stratified conditions, lungfish remained above the oxycline, rarely venturing into hypoxic waters, whereas during holomixis lungfish used a wider range of depths. Total volumetric space utilisation did not change significantly during stratified periods, but the distribution of space used changed, constrained by the oxycline. Despite N. forsteri having lungs to supplement oxygen requirements, the presence of a hypoxic zone constrained the core (50% 3-D-KUD) volumetric space used by lungfish to <1.6% of the total available space of the study area. With increasing demand for new impoundments in many tropical and subtropical regions, the present study provides insights to how air-breathing fish species may respond to altered riverine conditions from impoundments.

What is the most efficient respiratory organ for the loricariid air-breathing fish Pterygoplichthys anisitsi, gills or stomach? A quantitative morphological study

The purpose of the present study was to evaluate the morphometric respiratory potential of gills compared to the stomach in obtaining oxygen for aerobic metabolism in Pterygoplichthys anisitsi, a facultative air-breathing fish. The measurements were done using stereological methods. The gills showed greater total volume, volume-to-body mass ratio, potential surface area, and surface-to-volume ratio than the stomach. The water–blood diffusion barrier of the gills is thicker than the air–blood diffusion barrier of the stomach. Taken together, the surface area, the surface-to-volume ratio and the diffusion distance for O2 transfer from the respiratory medium to blood yield a greater diffusing capacity for gills than for the stomach, suggesting greater importance of aquatic respiration in this species. On the other hand, water breathing is energetically more expensive than breathing air. Under severe hypoxic conditions, O2 uptake by the stomach is more efficient than by the gills, although the stomach has a much lower diffusing capacity. Thus, P. anisitsi uses gills under normoxic conditions but the stomach may also support aerobic metabolism depending on environmental conditions.

High blood oxygen affinity in the air-breathing swamp eel Monopterus albus

The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O2 transport system to secure adequate O2 delivery to the respiring tissues. We therefore investigated O2 binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O2 equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O2 binding properties. The blood of M. albus has a high O2 carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O2 with an unusually high affinity (P50=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O2 affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue-1, respectively). We suggest that the high Hct and high blood O2 affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O2 flux from the blood to the muscles.

Air breathing in Magadi tilapia Alcolapia grahami, under normoxic and hyperoxic conditions, and the association with sunlight and reactive oxygen species

Observations of the Magadi tilapia Alcolapia grahami in hot, highly alkaline Lake Magadi revealed that they air breathe not only during hypoxia, as described previously, but also during normoxia and hyperoxia. Air breathing under these latter conditions occurred within distinct groupings of fish (pods) and involved only a small proportion of the population. Air breathing properties (duration and frequency) were quantified from video footage. Air breathing within the population followed a diel pattern with the maximum extent of pod formation occurring in early afternoon. High levels of reactive oxygen species (ROS) in the water may be an irritant that encourages the air-breathing behaviour. The diel pattern of air breathing in the field and in experiments followed the diel pattern of ROS concentrations in the water which are amongst the highest reported in the literature (maximum daytime values of 2.53 – 8.10 μM H₂O₂). Interlamellar cell masses (ILCM) occurred between the gill lamellae of fish from the lagoon with highest ROS and highest oxygen levels, while fish from a normoxic lagoon with one third the ROS had little or no ILCM. This is the first record of air breathing in a facultative air-breathing fish in hyperoxic conditions and the first record of an ILCM in a cichlid species.

The effect of sustained hypoxia on the cardio‐respiratory response of bowfin Amia calva: implications for changes in the oxygen transport system

This study examined mechanisms underlying cardio-respiratory acclimation to moderate sustained hypoxia (6.0 kPa for 7 days at 22° C) in the bowfin Amia calva, a facultative air-breathing fish. This level of hypoxia is slightly below the critical oxygen tension (pcrit ) of A. calva denied access to air (mean ± s.e. = 9.3 ± 1.0 kPa). Before exposure to sustained hypoxia, A. calva with access to air increased air-breathing frequency on exposure to acute progressive hypoxia while A. calva without access to air increased gill-breathing frequency. Exposure to sustained hypoxia increased the gill ventilation response to acute progressive hypoxia in A. calva without access to air, regardless of whether they had access to air or not during the sustained hypoxia. Additionally, there was a decrease in Hb-O2 binding affinity in these fish. This suggests that, in A. calva, acclimation to hypoxia elicits changes that increase oxygen delivery to the gas exchange surface for oxygen uptake and reduce haemoglobin affinity to enhance oxygen delivery to the tissues.

Effects of nitrite exposure on functional haemoglobin levels, bimodal respiration, and swimming performance in the facultative air-breathing fish Pangasianodon hypophthalmus

In this study we investigated nitrite (NO 2 −) effects in striped catfish, a facultative air-breather. Fish were exposed to 0, 0.4, and 0.9 mM nitrite for 0, 1, 2, 4, and 7 days, and levels of functional haemoglobin, methaemoglobin (metHb) and nitrosyl haemoglobin (HbNO) were assessed using spectral deconvolution. Plasma concentrations of nitrite, nitrate, chloride, potassium, and sodium were also measured. Partitioning of oxygen consumption was determined to reveal whether elevated metHb (causing functional hypoxia) induced air-breathing. The effects of nitrite on maximum oxygen uptake (MO 2max) and critical swimming speed ( U crit) were also assessed. Striped catfish was highly tolerant to nitrite exposure, as reflected by a 96 h LC 50 of 1.65 mM and a moderate nitrite uptake into the blood. Plasma levels of nitrite reached a maximum after 1 day of exposure, and then decreased, never exceeding ambient levels. MetHb, HbNO and nitrate (a nitrite detoxification product) also peaked after 1 day and then decreased. Only high levels of nitrite and metHb caused reductions in MO 2max and U crit. The response of striped catfish contrasts with that seen in most other fish species and discloses efficient mechanisms of combating nitrite threats. Furthermore, even though striped catfish is an efficient air-breather, this species has the ability to sustain aerobic scope and swimming performance without air-breathing, even when faced with nitrite-induced reductions in blood oxygen carrying capacity. Our study is the first to confirm that high levels of nitrite and metHb reduce MO 2max and thereby aerobic scope, while more moderate elevations fail to do so. Further studies are needed to elucidate the mechanisms underlying the low nitrite accumulation in striped catfish.

Metabolism, nitrogen excretion, and heat shock proteins in the central mudminnow (Umbra limi), a facultative air-breathing fish living in a variable environment

The central mudminnow ( Umbra limi (Kirtland, 1841)) is a continuous, facultative air-breathing freshwater fish found in swamps of central Canada and northeastern USA. The first goal of this field and laboratory-based study was to characterize the physicochemical conditions of mudminnow habitat during the summer. Our second goal was to determine the metabolic, stress response, and nitrogen excretion strategies of this fish following variations in water temperature, dissolved oxygen, external ammonia, and short-term periods of air exposure. We report profound diurnal fluctuations in water temperature (13–31 °C), dissolved oxygen (2%–159% air saturation), and ammonia levels (10–240 μmol·L−1) in habitat of central mudminnow measured on three dates at six different sites over 24 h. The central mudminnow does not induce urea synthesis as a mechanism of ammonia detoxification, either in response to emersion (6 or 20 h) or elevated external ammonia (10 mmol·L–1). Acute exposure to high temperature (~31 °C), aquatic hypoxia, or air resulted in significant increases in blood glucose and liver heat shock protein (Hsp) 70 and hypoxia also caused an increased reliance on anaerobic metabolism. This is the first description of the heat shock response in a facultative air-breathing fish following either hypoxia or air exposure. These metabolic and molecular responses are part of a strategy that allows the mudminnow to thrive in extremely variable freshwater environments.

Response of mucous cells of the gills of traíra (Hoplias malabaricus) and jeju (Hoplerythrinus unitaeniatus) (Teleostei: Erythrinidae) to hypo- and hyper-osmotic ion stress

The mucous cells (MC) of traíra, Hoplias malabaricus, and jeju, Hoplerythrinus unitaeniatus, two ecologically distinct erythrinid species, were analyzed in fish exposed to deionized (DW) and high ion concentration (NaCl and Ca2+) water (HIW) during 15 days to evaluate the MC responses to ion challenge. MCs are localized in the leading and trailing edge and, interlamellar region of the gill filament epithelium but, in H. unitaeniatus, they are also found in the breathing or lamellar epithelium. MC density is lower in H. malabaricus, the exclusively water-breathing fish, than in H. unitaeniatus, a facultative air-breathing fish. The transference to DW or HIW did not change the MC density and surface area, excepting in H. malabaricus, in the first day of exposure to DW. A single MC containing three types of glycoproteins (neutral, acidic and sulphated) was identified in the gill epithelium of both, H. malabaricus and H. unitaeniatus. The amount (based on the intensity of histochemistry reaction) of these glycoproteins differed between the species and were altered after exposure to DW and HIW showing little adjustments in the amount of mucosubstances in the MC of H. malabaricus and reduction of acidic and sulphated glycoproteins in H. unitaeniatus. The decreasing of these glycoproteins in H. unitaeniatus reduced the mucus protection against desiccation of gill tissue when change the ion concentration in water.

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na +/K +-ATPase, H +-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC 50 value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na +/NH 4 +-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H +-ATPase and Na +/K +-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H +-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H +-ATPase and Rhcg1 to the similar non-Na +/K +-ATPase immunoreactive cell type would support a role for H +-ATPase in ammonia excretion via Rhcg by NH 4 + trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H +-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H +-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH 3 permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.